The LSB represents a new multidisciplinary research initiative focused on the immune system, with an emphasis on quantitative, systems-level, microscopic and macroscopic analysis and computer modeling of immune functions, integration of these modeling efforts with data sets derived from global analyses of cell components, and the development and application of advanced imaging methods to the analysis of immune responses in vivo in models systems, and ultimately, man. The program is an attempt to understand the engineering of the immune system from the biochemical through the organismal levels and to generate predictive models based on such understanding, including how the immune system interacts with pathogens. A key effort will be the implementation of the type of measurement rigor needed for effective modeling at all levels of immune organization, from the biochemical to the whole animal. The Immunology Team, which is now part of the Lymphocyte Biology Section, Laboratory of Systems Biology, has undertaken as a major project a top-down analysis of the immune and non-immune tissue response of mice to various strains of influenza virus. Highly standardized preparations of both mildly pathogenic (Tx91) and highly pathogenic (PR8) viruses have been used at varying infectious doses in a single inbred strain of mouse and several hundred highly qualified microarray transcriptional analyses have been conducted with RNA isolated from infected lung tissues of these mice at varying time points post-inoculation. Tissues from the infected animals have also been subjected to cell recovery and highly multiplexed flow cytometric analysis, along with automated image analysis for tracking of the extent of influenza infection of cells within the lung tissue. These studies revealed markedly enhanced recruitment of myeloid cell subpopulations, especially inflammatory monocytes and neutrophils, in lungs of animals infected with the pathogenic PR8 viruses. We then developed methods for 9+ color flow cytometric separation of hematopoietic cell subsets (lymphoid and myeloid) and non-hematopoietic cells from infected lungs, validated the separation methods as not contributing to substantial changes in RNA patterns, and completed acquisition of microarray data on these purified subpopulations to increase the resolution of the analysis. These data all point to a special role for myeloid cells in the difference pathogenicity of the two primary strains of virus being studied. A variety of informatic tools have been employed to organize and analyze the emerging data sets, especially a k-means clustering algorithm that allows study of the RNA response in terms of co-regulated gene sets with linked function (modules). Such modular gene set analysis has uncovered marked differences in transcriptional responses with the PR8 vs. Tx91 strains of virus that can be assigned to specific biological processes. Principal component analysis (PCA) has shown that the transcriptional responses to infection results in cell-type specific changes that largely reflect cell recruitment into the lungs and also context (infection-type) specific changes within the cell-specific gene sets. Furthermore, gene sets related to inflammatory responses are the major component associated with lethality, whereas anti-viral gene sets are similar with both the low and high pathogenicity viruses. A positive feedback pathway involving virus-induced chemokine production that facilitates recruitment of myeloid cells to the lungs then leads to further recruitment upon exposure to virus. Finally, image analysis revealed that the highly pathogenic virus, despite a similar plaque titer to the non-pathogenic strain, spreads more extensively in the large airways and gas exchange components of the lungs. Together, these data suggest that uninterrupted amplification of myeloid cell recruitment and inflammatory cytokine production induced by rapidly spreading virus plays a key role in pathogenic infections. In support of this model, attenuating but not eliminating myeloid cell recruitment using depleting antibodies or myeloid cell-selective KO of Hif1a rescues mice from early lethality of PR8 infection, whereas total depletion accelerates disease due to a lack of basal innate immunity. Thus, this study has uncovered a core feedback circuit involving innate inflammation that drives early lethality in influenza infection and provides new targets for intervention in this disease. A second major project involves use of the emerging tools of systems biology to investigate the unexplored roles of many NLRs, a family of sensors found in key immune cells that provide a highly effective first line of defense against infection. In one part of this project, we have created constitutively active versions of many NLRs through truncation of the ligand-sensing LRR domain, expressed these activated forms using tet regulated lentiviruses, and conducted extensive transcriptional profiling of the induced cells. These data are being mined for information on functional biologic pathways activated by these molecules, providing clues to the signaling pathways utilized by the NLR to control cell function. In the course of the above informatic analysis, we also detected unexpected putative organellar localization signals in several NLRs, especially NLRP3, the main component of the best-characterized inflammasome. In following up this observation, we discovered that NLRP3 associates with mitochondria via the adapter MAVS, previously known solely for its involvement in type 1 interferon responses to viral RNA sensed by RIG-I. Using a combination of advanced imaging methods, biochemistry, molecular biology, and in vivo experimentation, we have determined that an N-terminal sequence outside the pyrin domain of NLRP3 mediates physical association between this NLR and MAVS, that MAVS recruitment to mitochondria plays a crucial amplifying role in NLRP3 inflammasome function leading to IL-1b and not type 1 interferon production, and that this activity is critical for NLRP3 inflammasome action during a model inflammatory process, acute tubular necrosis initiated by folic acid administration. These unexpected findings provide a mechanism for mitochondrial localization of NLRP3 and reveal a novel role for MAVS as a mediator of NLRP3 inflammasome activation.